LABIS IA P U MILA

FIE L D O F INV E NTION

T he present invention relates to a method for producing demethylbelamcandaquinone B from Labisia pumila. More particularly, the present invention relates to a method for producing demethylbelamcandaquinone B from Labisia pumila and use thereof. BAC KG R OU ND O F TH E INVE NTIO N

Labisia pumila, also known as Kacip Fatimah, is a small woody and leafy herbal plant that can be found in the shade of forest floors. Labisia pumila is widely used by traditional practitioners in S outh E ast Asia as a remedy for involution of birth channel, delay fertility and to regain body strength after childbirth.

Labisia pumila consists of several bioactive compounds with various biological activity and health benefits. Efforts in isolating the bioactive compounds of Labisia pumila have revealed healing properties that extends beyond fertility and post-partum care. An example of a study on the isolation of bioactive compounds of Labisia pumila and their biological activity was reported by Dianita et al (201 5).T he prior art reported a method for isolating demethylbelamcandaquinone B from Labisia pumila var. alata plant comprising the steps of providing dried and grounded Labisia pumila var. alata whole plant, obtaining an extract from the dried and grounded whole plant using n- hexane, subjecting the n-hexane extract to liquid chromatography with a mobile phase consisting of n-hexane and ethyl acetate and purifying the resulting fraction using liquid chromatography with a mobile phase consisting of dichloromethane and ethyl acetate to obtain demethylbelamcandaquinone B. T he isolated demethylbelamcandaquinone B compound was shown to have cardioprotective effects towards isoproterenol- induced myocardial infraction in rats. T hus, demethylbelamcandaquinone B could potentially be used to develop cardioprotective drugs.

With the increasing discovery of healing properties of Labisia pumila, there is a growing demand for its bioactive compounds. T herefore, there is a need for improvements to the existing method of isolating demethylbelamcandaquinone Bfrom Labisia pumila in order to increase its efficiency and yield. S U MMARY O F INVE NTIO N

T he present invention provides a method for producing demethylbelamcandaquinone B from Labisia P umila. Initially, Labisia P umila plant material is dried (step 101 ). T hen, the dried plant material is extracted using water (step 102) and then filtered (step 103). The filtrate is then freeze dried (step 104) and extracted via reflux extraction in dichloromethane to obtain crude extract (step 105). T hen, dichloromethane is removed to further concentrate the crude (step 106). T he concentrated crude is then subjected to column chromatography, the mobile phase consisting of chloroform and ethyl acetate (step 107). The obtained fractions are subjected to column chromatography where the mobile phase consists of methanol and chloroform (step 108). F inally, purification of fractions was carried out in column chromatography consisting of chloroform and ethyl acetate as mobile phase to obtain demethylbelamcandaquinone B (step 109).

P referably, the step of water extraction of powdered plant material is conducted for 2 hours at approximately 60eC . The said ratio of water to powdered plant material is approximately 1 :30. P referably, the freeze drying step is carried out for approximately 4 hours.

P referably, the step of obtaining an extract from the freeze dried solid reflux using dichloromethane is conducted for approximately 4 hours at the temperature of 40eC .

Additionally, the ratio of freeze dried solid to dichloromethane is preferably at

1 :10.

P referably, the removal of dichloromethane from the crude extract to concentrate the extract is conducted by rotary evaporation at the temperature of approximately 40eC.

It is yet another objective of the present invention to provide a use for demethylbelamcandaquinone B in the pharmaceutical preparations. Additionally, demethylbelamcandaquinone B is used for the manufacture of a medicament for the treatment of osteoporosis.

P referably, the dosage of demethylbelamcandaquinone B for the treatment of osteoporosis ranges between 0.0008 to 0.0017mg/kg body weight/day.

B RIE F DE S C RIPTIO N O F T HE DRAWING S

T he accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

FIG . 1 illustrates a flowchart of a method for producing demethylbelamcandaquinone B from Labisia pumila according to an embodiment of the present invention. FIG S . 2(A-B) illustrate liquid chromatography-mass spectrometry (LC/MS ) spectra of Labisia pumila extract and demethylbelamcandaquinone B produced according to the method of FIG . 1.

FIG . 3 illustrates ^Ί Η Nuclear Magnetic R esonance (NMR ) spectrum of demethylbelamcandaquinone B produced according to the method of FIG . 1 .

FIG . 4 illustrates ¹³ C Nuclear Magnetic R esonance (NMR ) spectrum of demethylbelamcandaquinone B produced according to the method of FIG . 1 . FIG S . 5(A-B) illustrate graphs detailing concentration dependant responses of osteoblast cells exposed to Labisia pumila water extract produced according to the method of FIG . 1.

FIG . 6 illustrates a graph detailing concentration dependant responses of osteoblast cells exposed to dichloromethane extract produced according to the method of FIG . 1.

FIG S . 7(A-B) illustrate graphs detailing concentration dependant responses of osteoblast cells exposed to M- estradiol and demethylbelamcandaquinone B produced according to the method of FIG . 1 . D E S C RIPTIO N O F T H E P R E F E R R E D E MB O DIME NT

A preferred embodiment of the present invention will be described herein below with reference to the accompanying drawings. In the following description, well known functions or constructions are not described in detail since they would obscure the description with unnecessary detail.

It is an objective of this invention to provide a method for producing demethylbelamcandaquinone B from Labisia pumila. Demethylbelamcandaquinone B has the ability to improve proliferation of osteoblast cells, and thus, could be utilized in the manufacture of a medicament for the treatment of osteoporosis.

Osteoporosis is a disease of progressive bone loss due to the disruption of bone-building process. Postmenopausal women are most likely to develop the condition, but the disease also affects men and younger people across all genders. Major factors that can lead to the disease include aging, heredity, poor nutrition, smoking, excessive alcohol consumption and the use of some medications such as steroids.

Bones are living tissues that must be constantly rebuilt. Bone-building process is done in two phases. First, cells called osteoclasts have the job of getting rid of old weakened bone. They resorb old bone to make room for the creation of healthy strong bone. Then, new bone is created by osteoblasts in the second phase. Osteoblast cells are immature bone cells that have the ability to produce a matrix composed of collagen thatthen becomes mineralized. Osteoblasts are vital for increasing bone density. Bone mass is maintained by a balance between the activity of osteoblasts that form bone and osteoclasts that break it down. In osteoporosis, the net rate of bone resorption exceeds the rate of bone formation, resulting in a decrease in bone mass. Over time, the bones can become brittle and prone to fracture as the osteoclasts remove more bone than the osteoblasts can create. Thus, for the treatment of osteoporosis, it has been suggested the disease could be remedied by increasing the proliferation of osteoblast cells.

R eference is now made to FIG . 1 which shows a flowchart showing the steps involved in producing demethylbelamcandaquinone B from Labisia pumila. Initially, Labisia pumila plant material is dried as in step 101 . The plant material includes leaves, roots and stems of the Labisia pumila. T he Labisia pumila plant material is preferably cleaned prior to air drying at room temperature. P referably, the Labisia pumila plant material is air dried away from direct sunlight.

T hereon, an extract is obtained from the dried plant material using water extraction as in step 102. P referably, said dried plant material is cut into small sizes or ground into powder form in order to facilitate the extraction process. T he step of obtaining an extract from the dried plant material using water extraction is preferably conducted at a temperature ranging between 60 to 65 eC for a period of approximately 2 hours. P referably, the weight ratio of said dried plant material to said water is approximately 1 :30. S aid water can either be distilled water, deionized water or any other purified water. Water is chosen as the initial extraction solvent as water is effective in extracting bioactive compounds of Labisia pumila. Most bioactive compounds in Labisia pumila are polar compounds. Water is more effective in extracting polar compounds in comparison to other organic solvents due to its high polarity and short chain. F urthermore, the presence of hydroxyl group in water forms hydrogen bonding with the polar compounds, which further aids the extraction. T hen, the extract is filtered to obtain a filtrate as in step 103.

Next, the filtrate is freeze dried to obtain a freeze-dried solid as in step 104. P referably, the step of freeze drying is conducted for a period of approximately 4 hours. T he freeze drying step involves cooling, freezing and sub-cooling processes at a temperature ranging between -4 to 30 eC. Freeze drying is a dehydration process carried out at low-temperature operating conditions. In the absence of the water solvent deterioration, microbial activity and chemical reactions are greatly reduced or eliminated in order to give better quality extracts in comparison to extracts dehydrated by conventional methods.

T hereon, the freeze-dried solid is extracted using reflux extraction in dichloromethane to obtain a crude extract as in step 105. The freeze-dried solid is extracted using reflux extraction means that include, but is not limited to S oxhlet and Kumagawa extraction. P referably, the step of obtaining an extract from the freeze-dried solid using reflux extraction in dichloromethane is performed at a temperature of approximately 40 eC for a period of approximately 4 hours. The temperature for reflux extraction is determined according to the boiling point of dichloromethane solvent which is around 39.6 eC. P referably, the weight ratio of said crude extract powder to said dichloromethane is approximately 1 :10.

S oxhlet and Kumagawa extraction is used when the desired compound have limited solubility in a solvent and the impurity is insoluble in that solvent. During S oxhlet and Kumagawa extraction, the solvent is heated to reflux. The solvent vapour then travels up a distillation arm and a condenser, and floods into a chamber housing a thimble and the material to be extracted. The condenser ensures that any solvent vapour cools, and drips back down into the chamber housing the material to be extracted. T he chamber containing the material to be extracted slowly fills with warm solvent. S ome of the desired compound will then dissolve in the warm solvent. When the chamber is almost full, the chamber is automatically emptied by a siphon side arm, with the solvent running back down to the distillation flask. This cycle may be allowed to repeat many times, over hours or days. During each cycle, only a portion of the desired compound dissolves in the solvent. After many cycles a larger amount of the desired compound could be extracted. T hus by utilizing reflux extraction methods such as S oxhlet and Kumagawa extraction method, it is possible to increase the efficiency of isolating demethylbelamcandaquinone B from Labisia pumila. Another advantage of these extraction method is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. F urthermore, S oxhlet and Kumagawa extraction allow for unmonitored and unmanaged operation while efficiently recycling a small amount of solvent to dissolve a larger amount of the target compound, demethylbelamcandaquinone B.

T hen, dichloromethane (DC M) is removed from the crude extract to obtain a concentrated extract as in step 106. T he DC M is removed using means that include, but is not limited to rotary evaporation. R otary evaporation reduces the volume of DC M solvent by distributing it as a thin film across the interior of a vessel at elevated temperature and reduced pressure. The pressure that is used in rotary vapour is suitably in the range of 80 to 100 rpm. T his promotes the rapid removal of excess solvent from less volatile samples. P referably, rotary evaporation is conducted at a temperature of approximately 40 eC until a solid concentrated extract is obtained. DC M is removed so that the moderately polar solvent does not interfere with the subsequent chromatographic isolation. Storage of concentrated extract when not in use is preferably conducted at a temperature of approximately -20 eC .

Next, the concentrated extract is subjected to column chromatography using a mobile phase consisting of chloroform and ethyl acetate at a ratio of approximately 9:1 as in step 107. P referably, the stationary phase for said column chromatography is silica gel.

T hereon, the fraction obtained in step 107 is subjected to column chromatography using a mobile phase consisting of methanol and chloroform at a ratio of approximately 1 :99 as in step 108. P referably, the stationary phase for said liquid chromatography is crosslinked hydroxypropylated dextran.

F inally, the fraction obtained in step 108 is purified using column chromatography using a mobile phase consisting of chloroform and ethyl acetate at a ratio of approximately 9:10 to obtain demethylbelamcandaquinone B as in step 109. P referably, the stationary phase for said column chromatography is silica gel.

T he method of the present invention is further illustrated by the following examples. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be constructed as limiting the scope of the embodiment herein.

E xamples of the E xperiments are illustrated as follows:

P rocedures

These examples illustrate the production of demethylbelamcandaquinone B from Labisia pumila of the var. alata specie.

Initially, Labisia pumila plant material was cleaned, cut and air dried at room temperature away from direct sunlight. In the present experiment, both leaves and roots of the plant were chosen for extraction. However, the leaves and roots were separately extracted in order to examine their individual osteoblastic activity. T he dried plant material was then grinded into powder. T hen, the powdered plant material was extracted in distilled water at a temperature of 60 eC for 2 hours. T he ratio of powdered plant material to distilled water was 1 :30. 100 mL of the water extract was sampled at every 5 minutes in the first 40 minutes and at every 10 minutes in the following 80 minutes for further analysis. The resulting water extract was filtered in order to obtain a filtrate and to remove residual particles. The yield of water extract was 700 g.

F iltrate from Labisia pumila water extract was freeze-dried to remove distilled water and to produce a freeze-dried solid. Then, the freeze-dried solid was placed in a S oxhlet extractor partitioned with dichloromethane, DC M, in the order of increasing polarity. The weight ratio of the freeze-dried solid to DC M was 1 :10. The fraction was boiled for 4 hours at approximately 40 eC . The boiling temperature was determined according to the boiling point of DC M. As a result 200 g of DC M extract was obtained. T he resulting DC M extract was then dried using a rotary evaporator at approximately 40 eC to remove the DC M solvent and to obtain 1 .3 g of concentrated extract. T he concentrated extract in solid form was stored at a temperature of approximately -20 eC for future use.

Demethylbelamcandaquinone B was isolated from the concentrated extract using column chromatography. 1.3 g of the concentrate extract was dissolved in 1 mL of mobile phase chloroform and ethyl acetate at a ratio of 9:1 , and loaded onto a silica gel packed glass column. A gradient mobile phase composed of different ratios of chloroform and ethyl acetate were used to isolate demethylbelamcandaquinone B. 15 fractions were collected and the compound elution was analysed using thin layer chromatography (T LC). T LC was performed using a mobile phase consisting of chloroform and ethyl acetate at a ratio of 9:1 in order to identify demethylbelamcandaquinone B. T he fraction was dissolved in absolute methanol and applied onto silica gel coated-T LC plates. T hen, the T LC plates were sprayed with anisaldehyde-sulphuric acid reagent and heated at 100 eC for 5 minutes. T he presence of any bioactive compounds was monitored under visible light and ultraviolet lights at wavelengths 254 nm and 366 nm .T he presence of demethylbelamcandaquinone B in the fractions is confirmed by the appearance of a prominent orange spot on the T LC plate. According to the result of T LC analysis on each fraction, demethylbelamcandaquinone B was identified to be in F raction 8 (81 .9 mg). Fraction 8 was then subjected to column chromatography using a column that was packed with crosslinked hydroxypropylated dextran beads. 1 % methanol in absolute chloroform were utilized as the mobile phase. As a result 10 fractions were collected and the compound elution was monitored by T LC . Demethylbelamcandaquinone B was determined to be in F raction 9 and Fraction 10 (53.6 mg). Then, F raction 9 and F raction 10 were subjected to further purification in silica gel packed glass column using a mobile phase of chloroform and ethyl acetate at a ratio of 9:10. The final yield for demethylbelamcandaquinone B is 35.0 mg.

C haracterization

The isolation of demethylbelamcandaquinone B from Labisia pumila was confirmed using a liquid chromatography coupled to a mass spectrometer (MS ) with a capillary voltage of 4500 V and column temperature of 40 eC . S ample extracts were dissolved in methanol and filtered with nylon 0.45uM. Analytes separation was carried out on a Zorbax C 18 column (5¾n, 1 50¾m B 4.6¾m) with a gradient mobile phase containing water (solvent A) and acetonitrile (solvent B), each containing 0.1 % formic acid and 5 mM ammonium formate. T he gradient started at 10% to 90 % of solvent B from 0.01 minutes to 8 minutes. T he condition were held for 3 minutes before returning back to 10 % of solvent B in 0.1 minutes and re-equilibrated for 5 minutes. For mass spectrum analysis, negative ion ionization mode was used. S urvey scans were conducted from 50 to 1200 m/z with an acquisition rate of 4 spectra per second.

Nuclear Magnetic Resonance (NMR) spectrum for the isolated demethylbelamcandaquinone B was performed by dissolving the isolated compound in deuterated chloroform (C DC ) to obtain a total sample amount of 600 i L. The sample was then poured into an NMR tube and the spectrum was observed on the applied magnetic field.

Osteoblast viability assay

Bioreactivity of demethylbelamcandaquinone B isolated from Labisia pumila was studied on osteoblast cells. Murine preosteoblast (MC3T3-E 1 ) cells were grown in ME M (Minimum E ssential Medium) containing 10 % Fetal Bovine S erum(F BS ), 10,000 units/mL of streptomycin and 25 i g/mL of F ungizone. The ME M culture media was then changed to differentiation medium -ME M supplemented with 50 =g/mLof ascorbic acid (AA) and 10 mM <f -glycerophosphate (G P) to induce osteoblast differentiation. The MC3T3-E 1 cells were plated into a 96-well plate at a seeding density of 1 B 10 ⁴ cells/Well. The cells were incubated at 37eC in an atmosphere of 5 % C C overnight. Then, the medium was replaced with differentiation media containing various concentrations of Labisia pumila water extract, Labisia pumila DC M extract and isolated demethylbelamcandaquinone B and incubated for 24 hours and 72 hours at 37eC in 5% C O2 atmosphere. The water extract, DC M extract and isolated demethylbelamcandaquinone B was prepared in 1 mL dimethyl sulfoxide (DMS O) at a final culture concentration ranging in between 0.001 % to 0.1 %. Additionally, M- estradiol was used as positive control for comparing proliferation effect of the isolated demethylbelamcandaquinone B on MC3T3-E 1 cells. M- estradiol was chosen for comparison as it is a steroid and estrogen sex hormone known to be effective in preventing osteoporosis. The M- estradiol was prepared in ethanol at a final culture concentration of 0.05 %. After incubation, 20¾_ of MTS solution was added into each well and cells were further incubated for 2¾burs to form MTS formazan product. Thereon, the absorbance of MTS formazan product formed was measured at 490¾n using a micro plate reader. The results of this viability were used to determine the optimum dose of demethylbelamcandaquinone B needed for the treatment of osteoporosis. E ach experiment was carried out in triplicates with at least 3 independent cultures with comparable results. C ell viability percentages are reported as mean e standard error of mean (S E M) of the three experiments. C omparisons between groups were made to be statistically significant (P< 0.05). R esult and discussions

Identification of anti-osteoporotic compound, demethylbelamcandaquinone B, in Labisia pumila concentrated extract was performed using LC/MS analysis using water and acetonitrile, each containing 0.1 % formic acid and 5 mM ammonium formate, as the mobile phase. FIG . 2(A) shows the specific corresponding peaks and retention times of demethylbelamcandaquinone B compound present in the concentrated extract. F rom the spectrum it can be deduced that the anti-osteoporotic compound may be collected as pure fraction at retention times between 4 to 7 minutes.

Demethylbelamcandaquinone B compound produced according to the described method was subjected to mass spectrum analysis and the result is shown in FIG.2(B). The compound was characterized using its pseudo molecular weight Mass spectrum of the compound exhibit a peak at m/z 680.7, which can be ascribed to the molar mass of demethylbelamcandaquinone B with ammonium adduct. The molecular structure of the isolated compound, demethylbelamcandaquinone B, was further identified using ^Ί Η and ¹³ C NMR spectrometers. FIG. 3 and FIG. 4 show the ^Ί Η and ¹³ C NMR spectrum of demethylbelamcandaquinone B, respectively. The isolated compound was established to be demethylbelamcandaquinone B with a molecular formula of C43H66O5. A peak observed at m/z 663.4883 can be ascribed to the molecular ion [M+H] ⁺ peak of demethylbelamcandaquinone Β. ^Ί Η and ¹³ C NMR spectroscopic data corresponding to the isolated demethylbelamcandaquinone B are summarized in TABLE 1.

The result of NMR analysis for demethylbelamcandaquinone B is described as follows: Demethylbelamcandaquinone B with the physical appearance of orange and waxy in the amount of 25.0 mg was obtained; (C HCI3); UV (EtOH)≡ _ma x nm (log ): 213 (3.16), 275 (3.04); IR (ATR) ¾ _ax , cm ¹ : 3275, 2922, 2854, 1680, 1638, 1618, 1600, 1456, 1339, 1226, 1147, 1051, 847, 722; positive HRESI-MS m/z: 663.4883 [M+H] ⁺ (C43H66O5, MW 662.9811); ^" Ή-NMR (600 MHz; CDCI ₃ ) _H (ppm): 0.89 (6H, m, H-21, H- 21'), 1.33-1.18 (18H, m, H-8-H-14, H-19-H-20, Η-8'-Η-14', H-19'-H-20', overlapped), 2.01 (8H, m, H-15, H-18, H-15', H-18'), 2.18 (H, m, H-7) 2.35( m, H-7), 2.23 (2H, m, H- 7'), 3.85 (s, OMe), 5.34 (4H, m, H-16-H-17, H-16'-H-17'), 5.99 (brs, OH), 6.16 (d,J =1.8 Hz, H-6'), 6.29 (d, J =1.8 Hz, H-4'); ¹³ C-NMR (150 MHz; CDCI ₃ ) c (ppm): 14.0 (C -21, C-21'), 22.4 (C-20, C-20'), 26.9 (C-18, C-18'), 27.2 (C-15, C-15'), 28.2 (C-7), 33.5 (C- 7'), 29.0-30.0 (C-8 ^" C-14), (31.8 (C-19), 32.0 (C-19'), 56.3 (OMe), 129.9 (C-16-C-17, C-16'-C-17'), quinone ring; 182.2 (C-1), 146.9 (C-2), 141.0 (C-3),188.0 (C-4), 107.4 (C-5), 158.9 (C-6), benzene ring; 153.7 (C-1'), 112.2 (C-2'), 143.2 (C-3'), 108.2 (C-4'), 156.7 (C-5'), 100.9 (C-6').

TABLE 1

No. c (ppm) _H (ppm)(J=Hz)

1 182.2 -

2 146.9 -

3 140.9 -

4 187.9 - 5 107.4 5.99, s

6 158.9 -

O Me 56.3 3.85, s

7 ax 28.2 2.35, m

7 eq 28.2 2.18, m

8-14 30.0-29.0 1.43-1 .18, m

15 26.9 2.01 , m

16 129.8 5.34, m

17 129.9 5.34, m

18 27.2 2.01 , m

19 31.8 1.43-1.18,m

20 22.4 1 .33, m

21 14.0 0.89, m

r 153.7 -

OH 5.93, br s

2' 1 12.2 -

3' 143.2 -

4' 108.2 6.29,d 0 =1.8)

5' 156.7 -

OH 6.09, br s

6' 100.9 6.16,d (J =1.8)

T 33.5 2.23, m

8'-14' 30.0-29.0 1.43-1 .18, m

15' 27.2 2.01 , m

16' 129.9 5.34, m

17' 129.9 5.34, m

18' 27.2 2.01 , m

19' 31.9 1.43-1 .18, m

20' 22.4 1.33, m

21 ' 14.2 0.89, m

T he concentration dependant responses of osteoblast cells exposed to water extract of Labisia pumila leaves and roots are shown in FIG . 5(A) and FIG . 5(B ), respectively. Osteoblast cells were exposed to Labisia pumila water extract at the concentrations of 5, 10, 25, 50, 100, 250, 500, 1000 and 2500 ι g/mL for 24 and 72 hours. It can be observed that both leaf and root extracts produced higher osteoblast cell viability percentage after 72 hour exposure in comparison to 24 hour exposure. T herefore, it can be concluded that both leaves and roots of Labisia pumila possess active osteoblast proliferation activity. T he findings further showed that cells exposed to leaf extract demonstrated better osteoblast cell viability in comparison to cells exposed to root extract As shown in FIG . 5(B), cell viability percentage dropped significantly to 0 % as the concentration of the root extract is increased to 500 ι g/mL. However for leaf extract shown in FIG . 5(A), a gradual decrease in cell viability is observed, before dropping to below 0 % after the cells were exposure to 2500 ι g/mL leaf extract It can be deduced that the leaves of Labisia pumila contain more of the active compounds having an active osteoblast proliferation activity than the roots of Labisia pumila.

R eferring now to FIG . 6, there is shown a graph detailing concentration dependant responses of osteoblast cells exposed to DC M extract Osteoblast cells were exposed to Labisia pumila DC M extract at the concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 ι g/mL for 24 and 72 hours. T he DC M extract used was further extracted from Labisia pumila leaf extract. Even at a lower concentrations, the cell viability percentages observed for DC M extract are significantly higher than the water extracts. T his finding shows that the DC M extract of the leaves contains more of the active compounds responsible for osteoblast proliferation activity than the water extract of the leaves. It is postulated that the step of extracting Labisia pumila leaf using reflux extraction in DC M solvent had improved the isolation of target compound demethylbelamcandaquinoneB.

R eferring now to FIG . 7(A) and FIG . 7(B), there is shown the concentration dependant responses of osteoblast cells exposed to M- estradiol and isolated demethylbelamcandaquinone B, respectively. Osteoblast cells were exposed to the compounds at concentrations of 0.02, 0.04, 0.06, 0.08, 0.1 , 0.2, 0.4, 0.6, 0.8 and 1 I g/mL for 24 and 72 hours. Interestingly, isolated demethylbelamcandaquinone B alone exhibited increased percentage of cell viability even at significantly lower concentrations than the water extracts and the DC M extracts. It can be deduced that demethylbelamcandaquinone B is on one of the active compounds in Labisia pumila extract that is responsible for the observed osteoblast proliferation activity. F urthermore, the use of demethylbelamcandaquinone B without the presence of other active compounds of the extract exhibited impressive osteoblast proliferation activity. Osteoblastic activity of demethylbelamcandaquinone B was compared with the positive control, M- estradiol. FIG . 7(A) and FIG . 7(B) show the percentage viability of cells exposed to17-<f estradiol is comparable to demethylbelamcandaquinone B at concentrations lower by 10 folds. M- estradiol is known to be effective in preventing osteoporosis. T hus, is it of no surprise that the osteoblast proliferation activity of M- estradiol is superior to that demethylbelamcandaquinoneB.

As a conclusion, the method for producing demethylbelamcandaquinone B from Labisia pumila as described was efficient at producing a good yield of the target compound. The above results demonstrate osteoblast proliferation activity of demethylbelamcandaquinone B. As previously discussed, osteoporosis disease is a condition brought upon by an imbalance between the activity of osteoblasts that form bone and osteoclasts that break it down. The isolated demethylbelamcandaquinone B could be used in the treatment of osteoporosis as the compound have been shown to increase the proliferation of osteoblast cells.

Isolated demethylbelamcandaquinone B compound produced according to the previously described method may be used in pharmaceutical preparations or medicaments for the treatment of osteoporosis. Additionally, the isolated compound may be used in a composition or in combination with carriers for ease of administration that is not limited to topical, enteral or parenteral delivery. T he dosage of demethylbelamcandaquinone B for the treatment of osteoporosis may range between 0.0008 to 0.0017 mg/kg body weight/day. While embodiments of the invention have been illustrated and described, it is not intended that these embodiments illustrate and describe all possible forms of the invention. Rather, the words used in the specifications are words of description rather than limitation and various changes may be made without departing from the scope of the invention.

R E F E R E NC E

Dianita R, J antan I, Amran AZ, J alii J , :P rotective Effects of Labisia P umila Var. Alata on Biochemical and Histopathological Alterations of Cardiac Muscle C ells in Isoproterenol-Induced Myocardial Infarction R ats ^" . MOLE C U LE S ., 201 5, 20(3), 4746- 63.